Differential Regulation of Cellular Senescence and Differentiation by Prolyl Isomerase Pin1 in Cardiac Progenitor Cells

Autologous c-kit⁺ cardiac progenitor cells (CPCs) are currently used in the clinic to treat heart disease. CPC-based regeneration may be further augmented by better understanding molecular mechanisms of endogenous cardiac repair and enhancement of pro-survival signaling pathways that antagonize senescence while also increasing differentiation. The prolyl isomerase Pin1 regulates multiple signaling cascades by modulating protein folding and thereby activity and stability of phosphoproteins. In this study, we examine the heretofore unexplored role of Pin1 in CPCs. Pin1 is expressed in CPCs in vitro and in vivo and is associated with increased proliferation. Pin1 is required for cell cycle progression and loss of Pin1 causes cell cycle arrest in the G₁ phase in CPCs, concomitantly associated with decreased expression of Cyclins D and B and increased expression of cell cycle inhibitors p53 and retinoblastoma (Rb). Pin1 deletion increases cellular senescence but not differentiation or cell death of CPCs. Pin1 is required for endogenous CPC response as Pin1 knock-out mice have a reduced number of proliferating CPCs after ischemic challenge. Pin1 overexpression also impairs proliferation and causes G₂/M phase cell cycle arrest with concurrent down-regulation of Cyclin B, p53, and Rb. Additionally, Pin1 overexpression inhibits replicative senescence, increases differentiation, and inhibits cell death of CPCs, indicating that cell cycle arrest caused by Pin1 overexpression is a consequence of differentiation and not senescence or cell death. In conclusion, Pin1 has pleiotropic roles in CPCs and may be a molecular target to promote survival, enhance repair, improve differentiation, and antagonize senescence.     

Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca²⁺. The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCeδB are resistant to oxidative stress induced by hydrogen peroxide (H₂O₂) relative to CPCe, whereas knockdown of CaMKIIδB resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and α-smooth muscle actin in CPCeδB compared with CPCe, suggesting increased differentiation. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages.     

Control of mouse myoblast commitment to terminal differentiation by mitogens

Regulation of the transition of mouse myoblasts from proliferation to terminal differentiation was studied with clonal density cultures of a permanent clonal myoblast cell line. In medium lacking mitogenic activity, mouse myoblasts withdraw from the cell cycle, elaborate muscle-specific gene products, and fuse to form multinucleated myotubes. Addition of a purified mitogen, fibroblast growth factor, to mitogen-depleted medium stimulates continued proliferation and prevents terminal differentiation. When mitogens are removed for increasing durations and then refed, mouse myoblasts irreversibly commit to terminal differentiation: after 2–4 h in the absence of mitogens, myoblasts withdraw from the cell cycle, elaborate muscle-specific gene products, and fuse in the presence of mitogens that have been fed back. Population kinetics of commitment determined with ³H-thymidine labeling and autoradiography suggest the following cell-cycle model for mouse myoblast commitment: (1) if mitogens are present in the extracellular environment of myoblasts in G₁ of the cell cycle, the cells enter S and continue through another cell cycle; (2) if mitogens have been absent for 2 or more hours, cells in G₁ do not enter S; the cells commit to differentiate, permanently withdraw from the cell cycle (will not enter S if mitogens are refed), and they subsequently elaborate acetylcholine receptors and fuse (even if mitogens are refed); (3) cells in other phases of the cell cycle continue to transit the cell cycle in the absence of mitogens until reaching the next G₁. The commitment kinetics and experiments with mitotically synchronized cells suggest that the commitment “decision” is made during G₁. Present results do not, however, exclude commitment of some cells in other phases of the cell cycle.     

Acidic Nucleoplasmic DNA-binding Protein (And-1) Controls Chromosome Congression by Regulating the Assembly of Centromere Protein A (CENP-A) at Centromeres

The centromere is an epigenetically designated chromatin domain that is essential for the accurate segregation of chromosomes during mitosis. The incorporation of centromere protein A (CENP-A) into chromatin is fundamental in defining the centromeric loci. Newly synthesized CENP-A is loaded at centromeres in early G₁ phase by the CENP-A-specific histone chaperone Holliday junction recognition protein (HJURP) coupled with other chromatin assembly factors. However, it is unknown whether there are additional HJURP-interacting factor(s) involving in this process. Here we identify acidic nucleoplasmic DNA-binding protein 1 (And-1) as a new factor that is required for the assembly of CENP-A nucleosomes. And-1 interacts with both CENP-A and HJURP in a prenucleosomal complex, and the association of And-1 with CENP-A is increased during the cell cycle transition from mitosis to G₁ phase. And-1 down-regulation significantly compromises chromosome congression and the deposition of HJURP-CENP-A complexes at centromeres. Consistently, overexpression of And-1 enhances the assembly of CENP-A at centromeres. We conclude that And-1 is an important factor that functions together with HJURP to facilitate the cell cycle-specific recruitment of CENP-A to centromeres.     

Clusterin induces CXCR4 expression and migration of cardiac progenitor cells

Clusterin (CST) is a stress-responding protein with multiple biological functions, including the inhibition of apoptosis and inflammation and transport of lipids. It may also participate in cell traffic and migration. In the process of post-infarct cardiac tissue repair, stem cells migrate into the damaged myocardium under the influence of chemoattractive substances such as stromal cell-derived factor (SDF). This study aimed at testing whether CST enhances expression of stem cell homing receptor and migration of cardiac progenitor cells (CPCs). CPCs isolated from fetal canine hearts transduced by CST cDNA expressed high levels of CXCR4, a receptor for SDF-1. The transfected cells also showed an increased migratory response to SDF-1 stimulation. The SDF-1-mediated migration of the CST-expressing CPCs was attenuated by PI3 kinase inhibitor LY294002 but not by mitogen-activated protein/ERK kinase inhibitor PD98059. Analysis of cell cycle by flow cytometry revealed no significant difference in cell cycle between the transduced and control CPCs. Thus, CST expression may increase CPCs migration via increasing CXCR4 expression and SDF-1/chemokine receptor signaling in a PI3/Akt-dependent manner.     

CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.     

Xenopus HJURP and condensin II are required for CENP-A assembly

Centromeric protein A (CENP-A) is the epigenetic mark of centromeres. CENP-A replenishment is necessary in each cell cycle to compensate for the dilution associated to DNA replication, but how this is achieved mechanistically is largely unknown. We have developed an assay using Xenopus egg extracts that can recapitulate the spatial and temporal specificity of CENP-A deposition observed in human cells, providing us with a robust in vitro system amenable to molecular dissection. Here we show that this deposition depends on Xenopus Holliday junction-recognizing protein (xHJURP), a member of the HJURP/Scm3 family recently identified in yeast and human cells, further supporting the essential role of these chaperones in CENP-A loading. Despite little sequence homology, human HJURP can substitute for xHJURP. We also report that condensin II, but not condensin I, is required for CENP-A assembly and contributes to retention of centromeric CENP-A nucleosomes both in mitosis and interphase. We propose that the chromatin structure imposed by condensin II at centromeres enables CENP-A incorporation initiated by xHJURP.     

The mystique of irreversibility in cyanobacterial heterocyst formation: parallels to differentiation and senescence in eukaryotic cells

The formation of cyanobacterial heterocysts is unique in the prokaryotic world: it is the only irreversible collective process. This terminal differentiation resembles senescence and differentiation in the eukaryotic urkingdom. During their cell cycle eukaryotic cells at the restriction point may reversibly proceed from a vegetative phase (G1) into a quiescent state (G0), and then may irreversibly enter the way towards differentiated or senescent cells. In parallel, at commitment point 1 vegetative cells from filamentous cyanobacteria may reversibly form proheterocysts, and then may proceed irreversibly towards mature heterocysts at commitment point 2. While the signals paving the path for differentiation or senescence in eukaryotes are largely unknown, heterocyst development is clearly triggered by nitrogen starvation. The reasons for the irreversibility in both systems are poorly understood. We discuss these questions, especially in the light of recent advances in the molecular biology of cyanobacteria, with emphasis on self-stabilizing autocatalytic cycles.     

Cdk activity couples epigenetic centromere inheritance to cell cycle progression

Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We further show that the key CENP-A assembly factor Mis18BP1(HsKNL2) is phosphorylated in a cell cycle-dependent manner that controls its centromere localization during mitotic exit. These results strongly support a model in which the CENP-A assembly machinery is poised for activation throughout the cell cycle but kept in an inactive noncentromeric state by Cdk activity during S, G2, and M phases. Alleviation of this inhibition in G1 phase ensures tight coupling between DNA replication, cell division, and subsequent centromere maturation.     

8-Hydroxydeoxyguanosine induces senescence-like changes in KG-1, human acute myelocytic leukemia cell line

8-Hydroxydeoxyguanosine (oh⁸dG) treatment induced senescence-like changes in KG-1 cells, a human acute myelocytic leukemia cell line. The oh⁸dG-treated cells stained positive for senescence associated β-galactosidase (SA-β-galactosidase) and had enlarged cell shape, both of which are senescence indexes. The oh⁸dG-treated cells were also cell growth inhibited and arrested at G₁ in the cell cycle. The accumulation of cdk (cyclin dependent kinase) inhibitors, such as p16, p21, and p27, also implies that cellular senescence was induced in oh⁸dG-treated cells. However, these changes were not accompanied by cell differentiation or telomerase activity. Taken together, we conclude that oh⁸dG treatment of KG-1 cells induces cellular senescence.     

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.     

Msx2 alters the timing of retinal ganglion cells fate commitment and differentiation

Timing of cell fate commitment determines distinct retinal cell types, which is believed to be controlled by a tightly coordinated regulatory program of proliferation, cell cycle exit and differentiation. Although homeobox protein Msx2 could induce apoptosis of optic vesicle, it is unclear whether Msx2 regulates differentiation and cell fate commitment of retinal progenitor cells (RPCs) to retinal ganglion cells (RGCs). In this study, we show that overexpression of Msx2 transiently suppressed the expression of Cyclin D1 and blocked cell proliferation. Meanwhile, overexpression of Msx2 delayed the expression of RGC-specific differentiation markers (Math5 and Brn3b), which showed that Msx2 could affect the timing of RGCs fate commitment and differentiation by delaying the timing of cell cycle exit of retinal progenitors. These results indicate Msx2 possesses dual regulatory functions in controlling cell cycle progression of retinal RPCs and timing of RGCs differentiation.     

Biphasic incorporation of centromeric histone CENP-A in fission yeast

CENP-A is a centromere-specific histone H3 variant that is essential for kinetochore formation. Here, we report that the fission yeast Schizosaccharomyces pombe has at least two distinct CENP-A deposition phases across the cell cycle: S and G2. The S phase deposition requires Ams2 GATA factor, which promotes histone gene activation. In Deltaams2, CENP-A fails to retain during S, but it reaccumulates onto centromeres via the G2 deposition pathway, which is down-regulated by Hip1, a homologue of HIRA histone chaperon. Reducing the length of G2 in Deltaams2 results in failure of CENP-A accumulation, leading to chromosome missegregation. N-terminal green fluorescent protein-tagging reduces the centromeric association of CENP-A, causing cell death in Deltaams2 but not in wild-type cells, suggesting that the N-terminal tail of CENP-A may play a pivotal role in the formation of centromeric nucleosomes at G2. These observations imply that CENP-A is normally localized to centromeres in S phase in an Ams2-dependent manner and that the G2 pathway may salvage CENP-A assembly to promote genome stability. The flexibility of CENP-A incorporation during the cell cycle may account for the plasticity of kinetochore formation when the authentic centromere is damaged.     

Commitment to differentiation in Friend cells and initiation of globin mRNA synthesis occurs during the G1 phase of the cell cycle

We have measured the kinetics of specific globin mRNA and Friend virus (FV) RNA synthesis by hybridization to immobilized cDNA after induction of differentiation of two erythroleukemia cell lines (F4N, B8) by butyrate and Me₂SO. The induction with butyrate in these cell lines occurs very rapidly (16–24 h). Cell cycle analysis was made of the populations throughout induction by flow cytofluorometry. The kinetics of commitment of cell populations to terminal differentiation by butyrate was determined by removal of inducer at various times and scoring of benzidine staining cells (hemoglobin producing). In addition, the cell cycle dependence of commitment was determined by flow sorting out of G1 and S+G2 cells various times after addition of inducer and scoring benzidine-stained colonies after growth in methylcellulose. Cells exposed to inducer were also sorted by cell cycle phase using an elutriator rotor. The amount of globin mRNA synthesis in the different cell populations was then determined.

1. 1. It was found that an 8–12 h period in butyrate was required before (a) globin specific mRNA was synthesized; and (b) commitment to differentiation occurred. The time course of globin mRNA synthesis was positively correlated with G1 arrest, as has been also found by others.

2. 2. The increase of FV RNA synthesis was not found during G1 arrest. It occurred early and before commitment.

3. 3. Commitment of cells to irreversible differentiation upon butyrate induction occurs only during the G1 phase of the cell cycle.

4. 4. Globin mRNA synthesis occurs first only in G1 cells.

5. 5. Globin mRNA is synthesized later in all phases of the cell cycle.

These data suggest that (a) commitment to differentiation and globin mRNA accumulation are coupled; and (b) that both events occur only in G 1 cells after a pre-commitment phase of about 12 h.     

The effects of calcium channel blockade on proliferation and differentiation of cardiac progenitor cells

Cardiogenesis depends on a tightly regulated balance between proliferation and differentiation of cardiac progenitor cells (CPCs) and their cardiomyocyte descendants. While exposure of early mouse embryos to Ca²⁺ channel antagonists has been associated with abnormal cardiac morphogenesis, less is known about the consequences of Ca²⁺ channel blockade on proliferation and differentiation of CPCs at the cellular level. Here we showed that at embryonic day (E) 11.5, the murine ventricles express several L-type and T-type Ca²⁺ channel isoforms, and that the dihydropyridine Ca²⁺ channel antagonist, nifedipine, blunts isoproterenol induced increases in intracellular Ca²⁺. Nifedipine mediated Ca²⁺ channel blockade was associated with a reduction in cell cycle activity of E11.5 CPCs and impaired assembly of the cardiomyocyte contractile apparatus. Furthermore, in cell transplantation experiments, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs and cardiomyocytes) was associated with smaller graft sizes compared to vehicle treated control animals. These data suggest that intracellular Ca²⁺ is a critical regulator of the balance between CPC proliferation and differentiation and demonstrate that interactions between pharmacological drugs and transplanted cells could have a significant impact on the effectiveness of cell based therapies for myocardial repair.     

Apoptosis in proliferating, senescent, and immortalized keratinocytes.

Skin provides an attractive organ system for exploring coordinated regulation of keratinocyte (KC) proliferation, differentiation, senescence, and apoptosis. Our main objective was to determine whether various types of cell cycle arrest confer resistance to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report, simultaneous expression of cyclin-dependent kinase inhibitors (p15, p16, p21, and p27), a marker of early differentiation (keratin 1), mediators of apoptosis (caspases 3 and 8), and NF-kappaB were analyzed in three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express p16; 3) Abruptly induced growth arrest, confluency, and senescence pathways are associated with resistance to apoptosis; 4) The death-defying phenotype of KCs does not require early differentiation; 5) NF-kappaB is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-kappaB, and diminished capacity to respond to antiproliferative treatments, and they remain highly sensitive to apoptosis with cleavage of caspases 3 and 8. These data indicate that KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-kappaB but not early differentiation.     

Control of cell cycle timing during C. elegans embryogenesis

As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first 8 out of 10 rounds of embryonic cell division in Caenorhabditis elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P₃ (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.